CN107663512A - Leucoderma Autologous epidermis melanin transplanted cells moisture is into activating method - Google Patents

Leucoderma Autologous epidermis melanin transplanted cells moisture is into activating method Download PDF

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CN107663512A
CN107663512A CN201610613248.4A CN201610613248A CN107663512A CN 107663512 A CN107663512 A CN 107663512A CN 201610613248 A CN201610613248 A CN 201610613248A CN 107663512 A CN107663512 A CN 107663512A
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melanocyte
culture
autologous
leucoderma
melanin
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陈智峰
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Changsha Huashan Vitiligo Hospital Co Ltd
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Changsha Huashan Vitiligo Hospital Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0626—Melanocytes
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/105—Insulin-like growth factors [IGF]
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

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Abstract

Comprised the following steps the invention discloses one kind using leucoderma Autologous epidermis melanin transplanted cells moisture into activating method：The scalp containing hair follicle on a small quantity is taken from patients head using skin puncher, melanocyte in hair follicle is separated and carries out in vitro culture；The culture of melanocyte and freeze and recover；Melanocyte culture and amplification；Form and function and stabilization characteristics of genetics the sexual biology identification of melanocyte.The present invention is by obtaining melanocyte in autologous scalp hair follicles, cultured and amplified in vitro obtains a large amount of autologous melanocytes, and this melanocyte has the normal melanocyte function of skin and can normally produce dermal melanin, after being transplanted to area for treatment, the melanocyte lacked at skin lesion can be effectively made up, so as to reach the purpose for the treatment of leucoderma.Such a treatment method is different from traditional therapy, and autogenous cell is not in rejection, does not need the treatment of medication thing, no potential risk more.

Description

Leucoderma Autologous epidermis melanin transplanted cells moisture is into activating method
Technical field
The present invention relates to one kind to utilize autologous hair follicle melanin transplanted cells moisture into activating method.
Background technology
Leucoderma is a kind of skin disease of common posteriority depigmentation, and pathogenesis is complicated.The whole world about 1% to 2% population is just perplexed by leucoderma, though more to this sick treatment method at present, how undesirable curative effect is.At present relatively Effective therapy is autologous melanocyte transplantation treatment, by the melanocyte that is obtained is from autologous, will not be caused Repel；Also it can be directed to and treat large range of skin lesion patient, be expected to thoroughly change the treatment difficult point of leucoderma, but due to autologous Melanocyte is derived from skin, is faced with limited amount and separation is difficult, culture process is complicated, is not easy the defects of popularization.Though So method for truly having some quick separating cells on the market, but because its cell derived needs substantial amounts of skin, transplanted Cell is not fixed easily, is not easy to survive, and distribution is also uneven, and curative effect is still imprecise, and medical expense is higher, remains in this way The drawbacks of certain.
The content of the invention
The defects of in the presence of prior art, it is an object of the invention to provide one kind to utilize leucoderma Autologous epidermis Melanin transplanted cells moisture is into activating method.
One kind, into activating method, is comprised the following steps using leucoderma Autologous epidermis melanin transplanted cells moisture：
The scalp containing hair follicle on a small quantity is taken from patients head using skin puncher, melanocyte in hair follicle is separated and goes forward side by side Row in vitro culture；
The culture of melanocyte and freeze and recover：Take epidermis sample, remove and be divided into that 2 × 2cm's is small after hypodermis Piece, the digestion of 0.25% 4 DEG C of trypsase is overnight.Postdigestive epidermis is placed in MCDB153 nutrient solutions, blown and beaten into unicellular outstanding Liquid, counted after centrifugation.It is inoculated in by 4~6 × 106/ml density in φ=35mm or 60mm culture dishes, 2- is passed on after 2~3 weeks 5 generations, conventional method freeze, standby；
Melanocyte culture and amplification：The DMEM/F12 culture mediums containing IGF-I, EGF, hydrocortisone and FBS are prepared, will Cell is inserted in culture medium and cultivated, and condition of culture is 36-38 DEG C, 4-6%CO2Saturated humidity environment incubator, it is every to change one within 2-4 days Subculture.
Form and function and stabilization characteristics of genetics the sexual biology identification of melanocyte：It is dopa stain using specific stain The biological characteristics of the melanocyte of in vitro culture is carried out with the dyeing of de- melanocyte, ImmunohistochemistryMethods Methods and transmission electron microscopy Identification.As a result show, the 26S Proteasome Structure and Function of the melanocyte of in vitro culture keeps normal.
The present invention obtains a large amount of autologous melanin by obtaining melanocyte in autologous scalp hair follicles, cultured and amplified in vitro Cell, and this melanocyte has the normal melanocyte function of skin and can normally produce skin black Element, after being transplanted to area for treatment, the melanocyte lacked at skin lesion can be effectively made up, so as to reach the mesh for the treatment of leucoderma 's.Such a treatment method is different from traditional therapy, and autogenous cell is not in rejection, does not more need medication thing to control Treat, no potential risk.
Embodiment
With reference to embodiment, the present invention is further illustrated, but is not limited thereto.
Embodiment 1
The scalp containing hair follicle on a small quantity is taken from patients head using skin puncher, melanocyte in hair follicle is separated and goes forward side by side Row in vitro culture；
The culture of melanocyte and freeze and recover：Take epidermis sample, remove and be divided into that 2 × 2cm's is small after hypodermis Piece, the digestion of 0.25% 4 DEG C of trypsase is overnight.Postdigestive epidermis is placed in MCDB153 nutrient solutions, blown and beaten into unicellular outstanding Liquid, counted after centrifugation.It is inoculated in by 4~6 × 106/ml density in φ=35mm or 60mm culture dishes, 2- is passed on after 2~3 weeks 5 generations, conventional method freeze, standby；
The preparation of autologous hair follicle melanocyte：2 scalps containing hair follicle are taken using 2mm skin punchers, with 70% After alcohol-pickled, PBS liquid is rinsed, and sterile saline is rinsed until surface cleaning, immediately with addition penicillin (400U/mL), Streptomysin (400mg/mL) and Tylosin (250mg/mL) PBS liquid embathe 3 times, are then immersed in sterile 0.25% neutral protein In enzyme, 4 DEG C of digestion process 5h are put into new PBS liquid, and hair follicle is taken out under microscope as in culture medium, is slowly added dropwise 0.25% Trypsase, 37 DEG C be incubated 10min after remove unnecessary trypsase, the culture medium more renewed, 37 DEG C, 5%CO2Saturated humidity ring Cultivated under border, you can it was observed that cell separates.Prepare the DMEM/F12 containing IGF-I, EGF, hydrocortisone and 2%FBS Culture medium, cell is inserted in culture medium and cultivated, condition of culture is 37 DEG C, 5%CO2Saturated humidity environment incubator, change within every 3 days One subculture.It is less than 20cm for repaired area2Affected part, the cell of amplification in vitro completed to supply Clinical practice at 1 week.
The clinical test of melanocyte allograft
Prepare cell suspension：0.25% trypsase digests 37 DEG C of the melanocyte of culture 10 minutes, and it is small to add 10% Cow's serum MCDB153 nutrient solution stopped reactions.Cell suspension is combined in into centrifuge tube, 1500 revs/min centrifuge 5~10 minutes.With slow Fliud flushing D-Hanks liquid washs 3~4 times, adjusts cell density 2-3 × 105/mL, prepares cell suspension with standby.
Clinic trial, skin graft at stationary phase patient 23 64 is selected to carry out the allograft of melanocyte, Huan Zhepi Blister is inhaled at damage to prepare：The vavuum pump for being 80.0kPa with negative pressure draws diameter 0.8cm~1.0cm blister at lesions of patients, makes Table, corium separation.
Cell transplantation：Blister liquid is pumped with syringe, injects above-mentioned 0.2~0.3mL of cell suspension, wrapping is fixed.As a result show, Patient's inflammatory reaction such as follow-up in 2 weeks, no redness after culture MC transplanting, blister liquid absorbs at most of lesions of patients, and blister wall comes off, Pigment occurs；Follow-up to January~April, pigment is persistently present.Confirm that melanocyte allogeneic transplant treatments leucoderma can treat Intractable leucoderma, and effect is good.

Claims (3)

1. one kind, into activating method, is comprised the following steps using leucoderma Autologous epidermis melanin transplanted cells moisture：
Step 1, the scalp containing hair follicle on a small quantity is taken from patients head using skin puncher, separates melanocyte in hair follicle And carry out in vitro culture；
Step 2, the culture of melanocyte and freezes and recovers；
Step 3, melanocyte culture and amplification；
Step 4, form and function and stabilization characteristics of genetics the sexual biology identification of melanocyte.
2. Autologous epidermis melanin transplanted cells moisture according to claim 1 is into activating method, it is characterised in that the step Rapid two include following method, take epidermis sample, and 2 × 2cm small pieces, 0.25% trypsase 4 are divided into after removal hypodermis DEG C digestion overnight, postdigestive epidermis is placed in MCDB153 nutrient solutions, single cell suspension is blown and beaten into, is counted after centrifugation, by 4 ~6 × 106/ml density is inoculated in φ=35mm or 60mm culture dishes, and 2-5 generations are passed on after 2~3 weeks, and conventional method freezes, It is standby.
3. Autologous epidermis melanin transplanted cells moisture according to claim 1 is into activating method, it is characterised in that the step Rapid three include following method, prepare the DMEM/F12 culture mediums containing IGF-I, EGF, hydrocortisone and FBS, cell is inserted into training Support and cultivated in base, condition of culture is 36-38 DEG C, 4-6%CO2Saturated humidity environment incubator, it is every to change within 2-4 days a subculture.
CN201610613248.4A 2016-07-29 2016-07-29 Leucoderma Autologous epidermis melanin transplanted cells moisture is into activating method Pending CN107663512A (en)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106399225A (en) * 2016-08-17 2017-02-15 重庆市中医院 A clinical application based melanocyte culture method
CN108795842A (en) * 2018-05-04 2018-11-13 江苏大学 A kind of 3D suspensions induce method and the application of iPS Hemapoiesis autologous melanoma cells
CN111139217A (en) * 2020-01-20 2020-05-12 杭州协合医疗用品有限公司 Epidermal melanocyte separation culture and cryopreservation method based on clinical application and cryopreservation liquid of melanocytes
CN112280732A (en) * 2020-10-21 2021-01-29 成都博润白癜风医院有限公司 Suspension of external root sheath of hair follicle and preparation method thereof
CN112522180A (en) * 2020-12-28 2021-03-19 上海宜治生物科技有限公司 Human scalp hair follicle single cell suspension and preparation method and application thereof
CN115671030A (en) * 2021-07-21 2023-02-03 杭州协合医疗用品有限公司 Melanocyte-containing sodium hyaluronate hydrogel capable of being injected by water light needle, preparation method and application

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106399225A (en) * 2016-08-17 2017-02-15 重庆市中医院 A clinical application based melanocyte culture method
CN108795842A (en) * 2018-05-04 2018-11-13 江苏大学 A kind of 3D suspensions induce method and the application of iPS Hemapoiesis autologous melanoma cells
CN111139217A (en) * 2020-01-20 2020-05-12 杭州协合医疗用品有限公司 Epidermal melanocyte separation culture and cryopreservation method based on clinical application and cryopreservation liquid of melanocytes
CN112280732A (en) * 2020-10-21 2021-01-29 成都博润白癜风医院有限公司 Suspension of external root sheath of hair follicle and preparation method thereof
CN112280732B (en) * 2020-10-21 2024-05-24 成都博润白癜风医院有限公司 Hair follicle outer root sheath suspension and preparation method thereof
CN112522180A (en) * 2020-12-28 2021-03-19 上海宜治生物科技有限公司 Human scalp hair follicle single cell suspension and preparation method and application thereof
CN115671030A (en) * 2021-07-21 2023-02-03 杭州协合医疗用品有限公司 Melanocyte-containing sodium hyaluronate hydrogel capable of being injected by water light needle, preparation method and application

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